Colorectal cancer was the third-most common cause of deaths from cancer in the United States in 2013. This disease claims over 50,000 lives and afflicts over 140,000 people annually in the US, underscoring a clear and urgent need for better control of this incurable malignancy. The goal of this study is to attenuate oncogenic activities in colon cancer cells by inhibiting a key proliferatory signaling pathway mediated by the powerful mitogen lysophosphatidic acid (LPA). In colon cancer, LPA signals through the LPA2 receptor, which recruits to its C-terminus a scaffolding protein NHERF-2. This recruitment occurs though a PDZ domain of NHERF-2 (N2P2)-a ubiquitous protein module that recognizes C-termini of partner proteins. Our goal is to inhibit the NHERF-2:LPA2 complex, and with it LPA signaling, using designed peptides that associate tightly with N2P2. Complicating this approach is the recent discovery that another PDZ-containing protein, MAGI-3, competes with NHERF-2 for binding to LPA2 and alters the functional outcome. Binding of NHERF-2 to LPA2 increases oncogenic signaling, while binding of MAGI-3 (via its PDZ domain M3P6) decreases it, underscoring the need for selectivity in N2P2 targeting. We are uniquely capable of providing such selectivity using our computational technologies that enable us to target a single member in a family of domains. Thus, our central hypothesis is that peptides designed to inhibit N2P2 will down-regulate oncogenic LPA signaling and that our computational methodology will uncover efficacious inhibitors with high N2P2 affinity and excellent selectivity against M3P6 and other PDZ domains, in vitro and in cells. The study will test our hypothesis through three specific aims. Aim 1 will establish that peptides already designed to target N2P2 and not M3P6 down-regulate LPA signaling in colon cancer cells. Aim 2 will test the hypothesis that our computational technology can produce peptides selective for N2P2 across the PDZome by designing and biophysically characterizing such peptides. Finally, Aim 3 will use a proteomic/mass-spectrometry assay along with fluorescence correlation techniques to quantify the selectivity of our peptides within cells and link biophysical parameters with function. We expect to 1) validate N2P2 targeting as a viable strategy for reducing LPA oncogenicity, 2) establish the functional relevance of selectivity against M3P6 (Aim 1) and other PDZ domains (Aims 2, 3), and 3) produce reagents suitable to initiate the development of colon cancer therapeutics. Our goals fit well with the overall theme of iTarget centered on biomolecular targeting. Being a part of the iTarget will accelerate our work significantly. Interactions with Drs. Kettenbach and McLellan will provide critical guidance in our proteomic/MS experiments and structural characterizations, respectively. Strong mentoring by Drs. Madden and Gerber on career and research matters will ensure high competitiveness for external funding. VMIC and MTC cores will be invaluable for in-cell and biophysical experiments (VMIC) and for expression and purification of proteins (MTC). Finally, iTarget will provide for a stimulating intellectual environment in which our research will thrive.